Human embryonic stem cells (hESC) have an inexhaustible ability to divide and renew, and under the appropriate conditions, differentiate and change into any cell type in the body. This balance between pluripotency and self-renewal is a complex and carefully choreographed response of the hESC to local microenvironmental cues. Understanding the molecular regulators of this balance, and the various signals that are integrated by hESC to maintain their pluripotency and self-renewal characteristics are critical for the expansion and differentiation of hESC to specific cell types, which is the ultimate goal of regenerative medicine.

EphrinB2 and ephB4 belong to a large family of cell surface signaling molecules, so called receptor tyrosine kinases (RTKs), that mediate and transduce signaling cascades upon interaction with each other. Cell-cell contacts between ephrinB2 and ephB4 expressing cells provide guidance cues for cell migration and boundary formation in many developmental systems such as the formation of neurons and blood vessels. Importantly, ephrinB2 has been determined to be a molecular marker of “stemness” and is expressed in human embryonic stem cells, neural stem cells and hematopoietic stem cells. Its cognate receptor, EphB4, has also been shown to affect mouse ESC fate. Despite much evidence from model systems that ephrinB2/ephB4 axis may be intimately involved in ESC fate (survival, self-renewal, and pluripotency), this particular axis has not been carefully studied in human ESC.

EphrinB-ephB ligand-receptor interactions are promiscuous, and the lack of highly specific reagents to block cognate ephrinB2-ephB4 interactions has hampered studies into the role of this RTK axis in regulating hESC survival, pluripotency and differentiation. Intriguingly, the envelope protein from an exotic and highly lethal virus called Nipah virus, binds to ephrinB2 with high specificity. Using an arsenal of reagents based on engineered versions of this viral envelope protein, which retains the ephrinB2 binding properties without the virulence of the actual virus, we will interrogate the ephrinB2-ephB4 axis in regulating hESC fate. Extant data from murine ESC suggest that EphB4 activation (signaling) not only favors mesodermal differentiation (a germlayer that gives rise to blood cells, endothelial cells, and muscle cells), but that EphB4 inactivation may result in expansion of primitive hematopoietic (blood) stem cells (HSC) while maintaining their "stemness". Understanding the regulation of this signaling axis could improve the culture of hESCs and the efficiency of HSC lineage differentiation, both key barriers in the field.

Statement of Benefit to California:

Human embryonic stem cells (hESC) have the potential to be a game changer in the practice of medicine. This is due to their apparent inexhaustible ability to divide and renew, and under the appropriate conditions, differentiate and change into any cell type in the body. In theory, scientists could differentiate hESC into any desired cell type in the test tube and use them to replace the desired cell type that is defective or wanting in the patient. This is the promise of regenerative medicine. However, the translation of this technology to actual patient use depends on a better understanding of how pluripotency (the ability to develop into any cell type) is maintained, and the specific conditions under which a particular cell type can be differentiated. This proposal will benefit California by seeking a better understanding of these processes, which will bring us closer to realizing the dream of regenerative medicine, and fulfilling the intent of Proposition 71.

Our proposal seeks to understand how a particular set of cell surface signaling molecules interact, and how these interactions lead to the maintenance of pluripotency or how they may skew hESC towards differentiation to a specific cell lineage. These cell surface signaling molecules are called ephrinB2 and ephB4, and their cognate interactions are known to trigger a cascade of intracellular signals that then determine the fate of hESC. Despite tantalizing evidence from animal model systems that the ephrinB2/ephB4 axis is important for many aspects of ESC fate, it has been difficult to interrogate this axis in human ESC biology due to the lack of highly specific reagents that can block this axis. This proposal capitalizes on a tool that Nature has “unwittingly” provided from an unexpected source. The viral envelope protein from an exotic virus (called Nipah virus) can bind to ephrinB2 with extraordinarily high specificity and block its interaction with ephB4. Using an arsenal of reagents based on this viral envelope protein, we will properly interrogate the role of the ephrinB2/ephB4 axis in hESC fate and development. Understanding the regulation of this signaling axis could improve the culture, expansion, and efficiency of lineage differentiation of hESCs, all key barriers in the field.

Review Summary:

EXECUTIVE SUMMARY

This proposal is focused on the role of the ephrinB2/ephB4 signaling pathway in human embryonic stem cell (hESC) self-renewal, pluripotency and differentiation. Genetic analysis in mouse models suggests that ephrins are intimately involved in the survival, self renewal and differentiation of mouse ESCs. However, their functional role in hESCs has not been addressed due to a number of technical challenges. The applicant has developed novel tools to overcome these challenges and proposes to apply them in three Specific Aims. In Aim 1, the applicant proposes to inhibit ephrinB2/ephB4 signaling in hESCs by various methods and assess pluripotency, self renewal and survival. In Aim 2, the applicant will examine the role of ephrinB2/ephB4 signaling in embryoid body formation and germ layer commitment. Finally, in Aim 3, the applicant proposes to investigate whether ephrinB2/ephB4 interactions promote the differentiation of hESCs into hematopoietic stem cells (HSCs).

Reviewers agreed that the proposed research is highly significant. Insights into Ephrin/Eph signaling gleaned from these studies could lead to improved protocols for culture and directed differentiation of hESCs. Aim 3 will specifically address the differentiation of hESCs into HSCs and may lead to methods for in vitro expansion of these cells, an important but elusive goal of HSC research. Reviewers agreed that this proposal is also highly innovative. Ephrin/Eph signaling interactions are difficult to study, due to a lack of experimentally accessible biological reagents, and have not been well characterized in stem cells, particularly hESCs. The research plan employs a number of novel tools and reagents to manipulate ephrinB2/ephB4 signaling and thereby addresses previously intractable questions.

Reviewers found the experimental design to be logical, creative and clever. They noted that the research plan is carefully designed to give meaningful results. Reviewers found the preliminary data persuasive and supportive of the Aims. They were impressed that the investigators have developed tools to distinguish between ephrin positive and negative cells and to specifically perturb ephrinB2/ephB4 interactions. In general, reviewers found the research plan feasible and appreciated its discussion of necessary controls, potential pitfalls and alternative approaches.

The reviewers described the PI and research team as having outstanding credentials to carry out the proposed research. The PI has published extensively in the field of virology, including many papers in top-tier journals. Reviewers appreciated that the research team includes experienced collaborators with complementary expertise in the areas of lentiviral transduction, humanized mouse models and HSC differentiation.

Overall, reviewers were extremely impressed by the quality of this proposal. They praised its highly innovative approach and were confident that it would produce quality data with the potential for broad impact in the field.